Identification and complementation of frameshift mutations associated with loss of cytadherence in Mycoplasma pneumoniae.

Mycoplasma pneumoniae cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of hmw2 by Tn4001 results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in hmw2. In this study, the recombinant wild-type hmw2 allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I hmw2 allele did not restore cytadherence, consistent with a defect in hmw2 in this mutant. A frameshift was discovered in different oligoadenine tracts in hmw2 from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in hmw2. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.     

Attachment organelle formation represented by localization of cytadherence proteins and formation of the electron-dense core in wild-type and mutant strains of Mycoplasma pneumoniae

Cytadherence proteins of Mycoplasma pneumoniae are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division. The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy. In the class I-2 mutant, which has a frameshift mutation in the hmw2 gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing. However, foci for P90, P40, P30, and P65 were not observed in this mutant. In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused. In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell. These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65. The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core. Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain. HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3. These differences were more pronounced in cytadherence mutants. These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65.     

Deletion analysis identifies key functional domains of the cytadherence-associated protein HMW2 of Mycoplasma pneumoniae

Mycoplasma pneumoniae attachment to host cells requires biogenesis of a functional attachment organelle, including proper localization of the adhesion protein P1 to this structure. Mutations in the hmw2 gene result in the inability to cytadhere, failure to localize P1 to the attachment organelle, altered cell morphology and accelerated turnover of the cytadherence-associated proteins HMW1, HMW3 and P65. The hmw2 gene encodes HMW2 (190 kDa) and P28 (28 kDa), the latter apparently the product of internal translation initiation near the 3' end of the hmw2 coding region. Transformation of hmw2 mutant I-2 with recombinant wild-type hmw2 restores a wild-type phenotype. In the current study, a severely truncated hmw2 gene with an in frame internal deletion of 80% of the HMW2 coding region that leaves the P28-encoding region intact restored cytadherence to mutant I-2. Transformants produced the expected 38 kDa HMW2 derivative (HMW2Deltamid) at levels comparable to that of HMW2 in wild-type cells; like HMW2, HMW2Deltamid exhibited marked Triton X-100 insolubility. HMW3, P65 and P28 were fully restored, but not HMW1. These transformants were morphologically similar to wild-type M. pneumoniae but failed to localize P1 to the attachment organelle. Finally, a C-terminally truncated HMW2 derivative was partly Triton X-100 soluble and incapable of restoring HMW1, HMW3 and P65 to wild-type levels. These data are consistent with a model in which the C-terminal domain of HMW2 imparts normal localization to the protein, and this localization itself is required for productive interactions with downstream cytadherence-associated proteins. Furthermore, these results emphasize the association of HMW1 with P1 clustering.     

Localization of Mycoplasma pneumoniae cytadherence-associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure

The terminal organelle of the cell wall-less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane-bound extension of the mycoplasma cell possesses a characteristic electron-dense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence-accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody-based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild-type M. pneumoniae and the hmw2- mutant I-2. The fusion protein was produced in both backgrounds at wild-type levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I-2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electron-dense core of the terminal organelle.     

Loss of co-chaperone TopJ impacts adhesin P1 presentation and terminal organelle maturation in Mycoplasma pneumoniae

Mycoplasma pneumoniae is a wall-less human respiratory tract pathogen that colonizes mucosal epithelium via a polar terminal organelle having a central electron-dense core and adhesin-related proteins clustered at a terminal button. A mutant lacking J-domain co-chaperone TopJ is non-cytadherent and non-motile, despite having a core and normal levels of the major cytadherence-associated proteins. J-domain co-chaperones work with DnaK to catalyse polypeptide binding and subsequent protein folding. Here we compared features of the topJ mutant with other cytadherence mutants to elucidate the contribution of TopJ to cytadherence function. The topJ mutant was similar ultrastructurally to a non-cytadherent mutant lacking terminal organelle proteins B/C, including aberrant core positioning and cell morphology in thin sections, but exhibited a hybrid satellite growth pattern with features of mutants both having and lacking a core. Time-lapse images of mycoplasmas expressing a YFP fusion with terminal organelle protein P41 suggested that terminal organelle formation/positioning was delayed or poorly co-ordinated with cell growth in the absence of TopJ. TopJ required a core for localization, perhaps involving HMW1. P1 trypsin accessibility on other non-cytadherent mutants was significantly enhanced over wild type but unexpectedly was reduced with topJ mutant cells, suggesting impaired processing, translocation and/or folding of this adhesin.     

Mycoplasma pneumoniae J-domain protein required for terminal organelle function

The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Colonization of the respiratory epithelium requires proper assembly of a complex, multifunctional, polar terminal organelle. Loss of a predicted J-domain protein also having domains unique to mycoplasma terminal organelle proteins (TopJ) resulted in a non-motile, adherence-deficient phenotype. J-domain proteins typically stimulate ATPase activity of Hsp70 chaperones to bind nascent peptides for proper folding, translocation or macromolecular assembly, or to resolve stress-induced protein aggregates. By Western immunoblotting all defined terminal organelle proteins examined except protein P24 remained at wild-type levels in the topJ mutant; previous studies established that P24 is required for normal initiation of terminal organelle formation. Nevertheless, terminal organelle proteins P1, P30, HMW1 and P41 failed to localize to a cell pole, and when evaluated quantitatively, P30 and HMW1 foci were undetectable in >40% of cells. Complementation of the topJ mutant with the recombinant wild-type topJ allele largely restored terminal organelle development, gliding motility and cytadherence. We propose that this J-domain protein, which localizes to the base of the terminal organelle in wild-type M. pneumoniae , functions in the late stages of assembly, positioning, or both, of nascent terminal organelles.     

Characterization of a Mycoplasma pneumoniae hmw3 mutant: implications for attachment organelle assembly

The proteins required for adherence of the pathogen Mycoplasma pneumoniae to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.     

Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.     

Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence-associated protein HMW1 and identification of HMW4 as a product

Mycoplasma pneumoniae is a major cause of tracheobronchitis and pneumonia in older children and young adults. The lack of adequate tools for genetic analysis has hindered the elucidation of function and regulation of mycoplasma virulence determinants. We describe here the use of a transposon vector to deliver the cloned gene for the cytadherence-associated protein HMW1 in M. pneumoniae . A 4.95 kbp Bam HI fragment encoding all but the C-terminal end of HMW1 was cloned into a modified Tn 4001 and transformed into wild-type M. pneumoniae and into a non-cytadhering mutant lacking HMW1–HMW5. Southern blot hybridizations confirmed insertion of the transposon and the presence of both the resident and recombinant hmw1 alleles. Analysis by Western immunoblotting revealed a truncated HMW1 (HMW1') in the transformants, the level of HMW1' being dependent upon the orientation of the hmw1 gene in the transposon and the site of insertion. Similar expression patterns were noted in wild-type and mutant backgrounds. However, expression of wild-type levels of HMW1' in the mutant did not restore adherence. Finally, HMW4 and HMW1 were shown to be products of the same gene, HMW4 being a heat-modified derivative of HMW1.     

Use of fluorescent-protein tagging to determine the subcellular localization of mycoplasma pneumoniae proteins encoded by the cytadherence regulatory locus

Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins--P65, HMW2, P41, and P24--that are encoded in the crl operon (for "cytadherence regulatory locus"), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding M. pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.     

P65 truncation impacts P30 dynamics during Mycoplasma pneumoniae gliding

The cell wall-less prokaryote Mycoplasma pneumoniae is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.     

Stability of Mycoplasma pneumoniae cytadherence-accessory protein HMW1 correlates with its association with the triton shell

Mycoplasma pneumoniae adsorbs to host respiratory epithelium primarily by its attachment organelle, the proper function of which depends upon mycoplasma adhesin and cytoskeletal proteins. Among the latter are the cytadherence-associated proteins HMW1 and HMW2, whose specific roles in this process are unknown. In the M. pneumoniae cytadherence mutant I-2, loss of HMW2 results in accelerated turnover of HMW1 and other cytadherence-accessory proteins, probably by proteolysis. However, both the mechanism of degradation and the means by which these proteins are rendered susceptible to it are not understood. In this study, we addressed whether HMW1 degradation is a function of its presence among specific subcellular fractions and established that HMW1 is a peripheral membrane protein that is antibody accessible on the outer surfaces of both wild-type and mutant I-2 M. pneumoniae but to a considerably lesser extent in the mutant. Quantitation of HMW1 in Triton X-100-fractionated extracts from cells pulse-labeled with [(35)S]methionine indicated that HMW1 is synthesized in a Triton X-100-soluble form that exists in equilibrium with an insoluble (cytoskeletal) form. Pulse-chase analysis demonstrated that over time, HMW1 becomes stabilized in the cytoskeletal fraction and associated with the cell surface in wild-type M. pneumoniae. The less efficient transition to the cytoskeleton and mycoplasma cell surface in mutant I-2 leads to accelerated degradation of HMW1. These data suggest a role for HMW2 in promoting export of HMW1 to the cell surface, where it is stable and fully functional.     

Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.     

Phosphorylation of cytadherence-accessory proteins in Mycoplasma pneumoniae.

Attachment to host cells of the respiratory epithelium by Mycoplasma pneumoniae is a complex, multicomponent process, requiring a number of accessory proteins in addition to adhesins directly involved in receptor binding. In this study, protein phosphorylation of the cytadherence-accessory proteins HMW1, HMW2, and HMW4 of M. pneumoniae was examined using biochemical and immunological techniques. The initial indication of protein modification came from Western immunoblot analysis of the two-dimensional polyacrylamide gel electrophoresis (PAGE) profile of M. pneumoniae proteins, revealing multiple spots for both HMW1 and HMW4 that varied in pI but not in size. M. pneumoniae cultured in the presence of H3(32)PO4 exhibited numerous phosphorylated proteins as detected by sodium dodecyl sulfate-PAGE and autoradiography. These included proteins corresponding to HMW1, HMW2, and HMW4 in electrophoretic mobility. The Triton X-100 partitioning characteristics of these phosphorylated proteins was identical to that described previously for HMW1, -2, and -4. Furthermore, these protein bands were absent when a noncytadhering variant deficient in HMW1-5 was examined in the same manner. Finally, the availability of antiserum to HMW1 and -4 enabled us to confirm by radioimmunoprecipitation that HMW1 and HMW4 are phosphoproteins. Phosphoamino acid analysis of acid-hydrolyzed HMW1 and HMW2 identified primarily phosphothreonine and, to a lesser extent, phosphoserine in HMW1 and predominantly phosphoserine, with a trace of phosphothreonine, in HMW2. Neither protein contained phosphotyrosine. HMW1-HMW5 are components of a cytoskeleton-like structure in M. pneumoniae that is thought to function in cell division, changes in cell morphology, gliding motility, and the localization of adhesins in the mycoplasma membrane. Phosphorylation may regulate cytoskeleton dynamics involving these cytadherence-accessory proteins.     

The gene mpn310 (hmw2) from Mycoplasma pneumoniae encodes two proteins, HMW2 and HMW2-s, which differ in size but use the same reading frame

The gene mpn310 from Mycoplasma pneumoniae encodes the proteins HMW2 with a molecular weight of 215 621 and the smaller P28, here called HMW2-s. Because HMW2-s is not well defined, it was isolated from protein extracts of M. pneumoniae cells and its N-terminal end was determined by MS. HMW2-s starts with the methionine at the amino acid position 1620 of HMW2 and its residual sequence is identical to the last 198 amino acids of HMW2, predicting a molecular weight of 23 204. These results were confirmed by the comparative MS analysis of HMW2-s that had been synthesized in Escherichia coli . A precursor–product relationship between HMW2 and HMW2-s could be excluded, because HMW2-s can be translated from a specific mRNA starting within mpn310 . The conservation of an HMW2-s like protein in M. pneumoniae and Mycoplasma genitalium emphasizes its possible functional importance.     

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.     

HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae

The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.     

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11

Summary Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5–5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.     

Transposon mutagenesis identifies genes associated with Mycoplasma pneumoniae gliding motility

The wall-less prokaryote Mycoplasma pneumoniae, a common cause of chronic respiratory tract infections in humans, is considered to be among the smallest and simplest known cells capable of self-replication, yet it has a complex architecture with a novel cytoskeleton and a differentiated terminal organelle that function in adherence, cell division, and gliding motility. Recent findings have begun to elucidate the hierarchy of protein interactions required for terminal organelle assembly, but the engineering of its gliding machinery is largely unknown. In the current study, we assessed gliding in cytadherence mutants lacking terminal organelle proteins B, C, P1, and HMW1. Furthermore, we screened over 3,500 M. pneumoniae transposon mutants individually to identify genes associated with gliding but dispensable for cytadherence. Forty-seven transformants having motility defects were characterized further, with transposon insertions mapping to 32 different open reading frames widely distributed throughout the M. pneumoniae genome; 30 of these were dispensable for cytadherence. We confirmed the clonality of selected transformants by Southern blot hybridization and PCR analysis and characterized satellite growth and gliding by microcinematography. For some mutants, satellite growth was absent or developed more slowly than that of the wild type. Others produced lawn-like growth largely devoid of typical microcolonies, while still others had a dull, asymmetrical leading edge or a filamentous appearance of colony spreading. All mutants exhibited substantially reduced gliding velocities and/or frequencies. These findings significantly expand our understanding of the complexity of M. pneumoniae gliding and the identity of possible elements of the gliding machinery, providing a foundation for a detailed analysis of the engineering and regulation of motility in this unusual prokaryote.     

Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.